The present invention relates to a method for amplifying nucleic acid molecules and in particular, to a method for amplifying nucleic acid molecules of a single kind. The present invention also pertains to a method for synthesizing a protein using a cell-free protein-synthesis system. Moreover, the present invention further relates to a method for constituting a protein library using the foregoing methods for amplifying nucleic acid molecules and for synthesizing proteins.
To preferentially amplify nucleic acid molecules of a single kind out of many kinds of nucleic acid molecules, there has been adopted a method which makes use of a combination of a plasmid and a host microorganism such as Escherichia coli or yeast. This method makes use of such a characteristic of the plasmid that only one plasmid molecule is taken in a host microorganism such as E. coli or yeast and comprises the steps of introducing a plasmid into a host microorganism by the calcium chloride method or the electroporation method, isolating-proliferating the microorganism to give a colony of the microorganism containing nucleic acid molecules of a single kind and to thus amplify the nucleic acid molecules of a single kind.
However, for instance, the linkage of a desired nucleic acid molecule to a plasmid, the introduction of the plasmid into a host microorganism and the isolation and proliferation of the microorganism require very complicated operations and as a result, they require the use of an advanced technology and a great deal of expenses and time. In particular, DNA molecules of a single kind should be isolated from vast kinds of DNA molecules in the field wherein industrially useful proteins are produced by altering the base sequences of genetic DNA's coding for amino acid sequences, producing variant proteins whose amino acid sequences are changed by using the resulting DNA variants, but it is very difficult to practice such operations.
Moreover, in the cell-free protein-synthesis system, the operations required for the synthesis of proteins are quite simple as compared with the protein-synthesis system which makes use of, for instance, organisms or living cells such as cultured cells and for this reason, there has recently been desired for the development of such a cell-free system. In addition, the PCR (polymerase chain reaction) technique is an extremely effective and simple means for amplifying DNA genes. Under such circumstances, there has been desired for the development of a technique, which comprises the combination of these two techniques, for easily and directly synthesizing a large amount of proteins using the DNA's which code for the proteins and which are produced by the PCR technique without using any plasmid DNA.
There has already been known a cell-free protein-synthesis system using DNA's produced by the PCR technique (Proc. Natl. Acad. Sci. USA, 1997, 94, pp. 412-417). However, it has been difficult to synthesize a protein in a desired amount and therefore, it has been an important subject to improve the yield of proteins synthesized by this technique.